An individual needs to have a dynamic immune system that is able to adapt rapidly to respond adequately to potentially harmful microorganisms and to respond to the exposure of a highly diverse and continuously changing environment. Higher organisms have evolved specialized molecular mechanisms to ensure the implementation of clonally-distributed, highly diverse repertoires of antigen-receptor molecules expressed by cells of the immune system: immunoglobulin (Ig) molecules on B lymphocytes and T cell receptors on T lymphocytes. A primary repertoire of (generally low affinity) Ig receptors is established during B cell differentiation in the bone marrow as a result of rearrangement of germ line-encoded gene segments. Further refinement of Ig receptor specificity and affinity occurs in peripheral lymphoid organs where antigen-stimulated B lymphocytes activate a somatic hypermutation machinery that specifically targets the immunoglobulin variable (V) regions. During this process, B cell clones with mutant Ig receptors of higher affinity for the inciting antigen are stimulated into clonal proliferation and maturation into antibody-secreting plasma cells (reviewed in Berek and Milstein, 1987).
Recombinant DNA technology has been used to mimic many aspects of the processes that govern the generation and selection of natural human antibody repertoires (reviewed in Winter and Milstein, 1991; Vaughan et al. 1998). The construction of large repertoires of antibody fragments (such as Fab fragments or single chain Fv fragments, scFv's) expressed on the surface of filamentous phage particles and the selection of such phages by “panning” on antigens has been developed as a versatile and rapid method to obtain antibodies of desired specificities (reviewed in Burton and Barbas, 1994). A subsequent optimization of the affinity of individual phage antibodies was achieved by creating mutant antibody repertoires of the selected phages and sampled for higher affinity descendents by selection for binding to antigen under more stringent conditions (reviewed in Hoogenboom, 1994).
M13 and M13-derived phages (sometimes also called viruses) are filamentous phages that can selectively infect F-pili bearing (F+) Escherichia coli (E. coli) cells. The phage genome encodes 11 proteins, while the phage coat itself consists of five of these proteins: gene-3, -6, -7, -8 and -9 (g3, g6, g7, g8 and g9) proteins that are bound to and that protect the (circular) (circular) single stranded DNA (ssDNA) of the viral genome. The life cycle of the virus can be subdivided into different phases. After infection of an E. coli by a phage particle, the ssDNA of the virus becomes double stranded due to the action of a number of bacterial enzymes. The double stranded phage genome now serves as a template for the transcription and translation of all 11 genes located on the phage genome. Besides these protein-encoding regions, the phage genome contains an intergenic region: the F1-origin of replication initiation (F1-ORI). The DNA sequence of this F1-ORI can be divided in two separate subregions. One subregion is responsible for the initiation and termination of the synthesis of ssDNA via the so-called “rolling circle mechanism” and the other subregion is responsible for the packaging initiation of the formed circular ssDNA leading to the formation and release of new virus particles.
It has been shown that polypeptides, such as stretches of amino acids, protein parts or even entire proteins can be added by means of molecular genetics to the terminal ends of a number of particle coat proteins, without disturbing the functionality of these proteins in the phage life cycle (Smith, 1985; Cwirla et al. 1990; Devlin et al. 1990; Bass et al. 1990; Felici et al. 1993; Luzzago et al. 1993).
This feature enables investigators to display peptides or proteins on phages, resulting in the generation of peptide- or protein expression phage display libraries. One of the proteins that has been used in the art to fuse with polypeptides for phage display purposes, is the g3 protein (g3p), which is a coat protein that is required for an efficient and effective infectivity and subsequent entry of the viral genome into the E. coli cell.
For the production of phages that display polypeptides fused to the g3p coat protein, investigators in the art introduced a plasmid together with the phage genome in E. coli cells. This plasmid contains an active promoter upstream of an in-frame fusion between the g3 encoding gene and a gene of interest (X) encoding, for instance, polypeptides, proteins, antibodies or fragments such as Fab fragments or scFv's. The introduction of this plasmid together with the genome of the helper phage in an E. coli cell results in the generation of phages that contain on their coat either the wild type g3p from the viral genome, the fusion product g3p-X from the plasmid or a mixture of the two, since one phage particle carries five g3p's on its surface. The process of g3p or g3p-X incorporation is generally random. The presence of an F1-ORI sequence in the g3p-X expression vector (plasmid) misleads the phage synthesis machinery in such a way that two types of circular ssDNA are formed: one is derived from the genome of the phage and the other is derived from the expression vector. During the synthesis of new phages, the machinery is unable to distinguish the difference between these two forms of ssDNA resulting in the synthesis of a mixed population of phages, one part containing the phage genome and one part harboring the vector DNA. Due to these processes, the mixture contains at least some phages in which the phenotypic information on the outside (the g3p-X fusion protein) is conserved within the genotypic information inside the particle (the g3p-X expression vector). An infectious wild type phage and a phage carrying a fusion protein attached to g3p are depicted in FIG. 4. The art teaches that there are several problems that concern the use of these basic set-ups.
The high level of genotypic wild type phages in phage populations grown in bacteria that contain both the phage genome and the expression vector, compelled investigators to design mutant F1-ORI sequences in M13 genomes. Such mutant M13-strains are less effective in incorporating their genome in phage particles during phage assembly, resulting in an increased percentage of phages containing vector sequences when co-expressed. These mutant phages, such as the commercially available strains R408, VCSM13 and M13KO7, are called “helper phages.” The genome of these helper phages may contain genes required to assemble new (helper-) phages in E. coli and to subsequently infect new F-pili expressing E. coli. Both VCSM13 and M13K07 were provided with an origin of replication initiation (ORI) of the P15A type that results in the multiplication of the viral genome in E. coli. Moreover, the ORI introduction ensures that after cell division, the old and newly formed E. coli contains at least one copy of the viral genome.
It was suggested and finally proven by several investigators that the introduction of plasmids containing a g3p-scFv fusion product, together with the genome of the helper phages in E. coli cells, results in approximately 99% of newly formed phages that harbor the g3p-scFv fusion protein expression plasmid but, nevertheless, lack the g3p-scFv fusion on its surface (Beekwilder et al. 1999). The absence of g3p-X is a significant disadvantage in the use of display libraries for the identification of specific proteins or peptides, such as scFv's that bind to a target of interest (such as tumor antigens). It implies that in the case of phage display libraries, at least a 100-fold excess of produced phages must be used in an experiment in order to perform a selection with all possible fusion proteins present. The art teaches that this overload of relatively useless phages in an experiment leads to too many false positives. For instance, at least 1012 phages should be added to a panning experiment in order to have one copy of each possible fusion present in the experiment since such a library contains approximately 1010 different g3p-scFv fusions (1%). The phages in this approximate 1% express generally only 1 g3p-scFv fusion on their coat together with four normal g3p's (no fusions), while the rest of the helper phages (approximately 99%), express five g3p's and no g3p-scFv fusions. To ensure, theoretically, the presence of 100 copies of each separate fusion protein in a panning experiment, one needs to use approximately 1014 phages in such an experiment. Persons skilled in the art generally attempt to use an excess of at least 100-fold of each single unique fusion protein to ensure the presence of sufficient numbers of each separate fusion and to prevent losing relevant binders too quickly in first panning rounds. That number of phages (1014) is more or less the maximum of phage particles that one milliliter (ml) can hold. The viscosity of such a solution is extremely high and, therefore, relatively useless. Especially when ELISA panning strategies are used (in which the volume of one well is only 200 μl), such libraries cannot be used.
In addition to these problems, it is shown that, depending on the antigen, an average of one in every 107 phages will bind to the antigen due to a-specific binding. Generally, as mentioned, for the addition of 1012 scFv expressing input phages (1%) to a panning procedure, one has to add approximately 1014 phages (99% does not express a scFv fragment). It is generally assumed that from these 1012 phages, approximately 104 particles might be putatively interesting phages. However, the number of calculated background phages that are normally found by using libraries present in the art after one round of panning was approximately 106−107, while only a few of these phages appear to be relevant binders. This is one of the most significant problems recognized in the art: too many background phages show up as initial binders in the phage mix after the first round of panning, while only a few significant and interesting binders are present in this mix. So, the absolute number of isolated phages after one round of panning is clearly too high (106–107). Moreover, in subsequent rounds of panning, non-specific background phages also remain present. In libraries used in the art, most of these non-specific binders will amplify on bacteria to continue appearing in a second round of panning. Therefore, the art teaches that the background level of a-specifically binding phages and the total number of phages per ml in these types of libraries is unacceptably high and remains high during subsequent rounds of panning.
A possibility that was suggested by investigators in the art as a solution to the problem of obtaining too many background phages that lack a g3p-X fusion, was to remove the g3p-encoding gene entirely from the helper phage genome. In principle, this system ensures that during phage synthesis in an E. coli cell (that received the g3-less phage genome and a g3p-X fusion protein expression vector), only g3p-X proteins are incorporated in the newly formed phage coat. By doing so, each synthesized phage will express five copies of the g3p-X fusion product and hardly any phages are synthesized that express the g3p alone or that express less than five g3p-X fusions. R408-d3 and M13αD3 are two examples of g3-minus helper phages (Dueòas and Borrebaeck, 1995; Rakonjac et al. 1997). Because the genome of these phages in not capable of supporting g3p synthesis, phage particles that carry less than five g3p-X fusion proteins can hardly be formed or, if formed, are found to be non-infectious due to instability, since the art teaches that five g3p's are necessary to ensure a stable phage particle.
To produce helper phages that do not contain the g3 gene but that are, nevertheless, infectious, that can be used to generate libraries of phages that carry five g3p-X fusion proteins, and that lack phages with less than five g3p-X fusions, it was recognized in the art that an external source for g3p was required. Such a source can be a vector without F1-ORI but that, nevertheless, contains an active promoter upstream of the full open reading frame (ORF) of g3. One major problem that is recognized by persons skilled in the art is that after the generation step of producing newly formed helper phages lacking a g3 gene, the yield is dramatically low. In fact, the yield of all described systems is below 1010 phages per liter, meaning that for a library of 1010 individual clones, at least 100 liters of helper phage culture is necessary (NB: the helper phages need to be purified) in order to grow the library once. Thus, the art teaches that phage libraries generated with such low titers of helper phages are not useful for phage display purposes and that, therefore, these libraries cannot be used for panning experiments.
Phages that express deleted g3p's fused to heterologous proteins have also been generated. For the construction of most conventional Fab libraries and some scFv phage display libraries, the D1 domain, and parts of the D2 domain, were removed to ensure a shorter fusion protein, which was considered in the art as a product that could be translated easier than a full length g3p linked to a full length Fab fragment. The shorter g3p part would not prevent the generation of a viable and useful helper phage. Of course, such phages still depend on full length g3p's that are present on their surface next to the deleted g3p fusion with the Fab fragment for functional infectivity of E. coli cells. Also, phages that express deleted g3p's fused to ligand-binding proteins have been generated that depend on their infectious abilities on antigens that were fused to the parts of g3p that were missing from the non-infectious phage particle (reviewed by Spada et al. 1997). These particles depend for their infectivity on an interaction between the ligand-binding protein and their respective ligand.
The g3-minus helper phages R408-d3 and M13ΔD3 mentioned above, lack in their genome a bacterial ORI and a selection marker. The absence of a selection marker in the g3-minus genomes has a significant effect on the production scale of helper phages, because it results in an overgrowth of bacteria that do not contain the helper phage genome. It is known that bacteria grow slower when infected with the helper phage or virus. Therefore, bacteria that lack the phage genome quickly overgrow the other bacteria that do contain the genome. Another effect of the lack of an ORI or a selection marker is that g3-minus phage genomes cannot be kept in dividing bacteria during the production and expansion of phage display libraries. This is a very important negative feature because overgrowth of bacteria that lost the phage genome or that did never receive one, appear to have a growth advantage over bacteria that do contain the phage genome. In addition, of course, such “empty” bacteria are not capable of producing any phage and, as a result, the phage display vectors including fusion protein fragments in such lacking-bacteria helper phages are lost permanently.
As mentioned, the g3p's are thought to be essential for the assembly of stable M13-like phages and, because of their crucial role in infection, g3p's should be provided otherwise when g3-minus helper phages are to be generated. There is a prejudice in the art against making phage display libraries that lack g3p's because phages lacking g3p's are not stable. Rakonjac et al. (1997) constructed a VCSM13 g3-minus helper phage in parallel to a R408 g3-minus helper phage and used helper plasmids, with either the psp or the lac promoter upstream of a full length g3 sequence, to substitute g3 during helper phage synthesis (Model et al. 1997). However, the art teaches that the lac promoter has the disadvantage that it cannot be shut off completely, not even in the presence of high concentrations of glucose (3–5%) in the medium (Rakonjac and Model, 1998). An additional problem that is well known in the art is that even very low levels of g3p in E. coli can block infection of M13-like phages. Moreover, it has been shown that co-encapsidation of plasmids, together with the phage genome, can occur (Russel and Model, 1989; Krebber et al. 1995; Rakonjac et al. 1997). If co-encapsidation occurs with the lac-driven helper plasmid, it will compete with the lac-driven vectors used in the phage display, resulting in the efficient production of infectious phage particles that will not contain the g3p-X fusion product. Together, the art thus teaches that the lac promoter is not the best candidate promoter in the helper plasmid system. The psp promoter has the advantage of being relatively silent in E. coli until infection (Rakonjac et al. 1997). Upon M13-class phage infection, the psp promoter becomes activated and now the helper plasmid will produce g3 proteins. However, the disadvantage of this promoter is that the level of RNA production cannot be regulated with external factors, but has to be regulated by either mutating (and changing the activity of) the promotor, changing the ribosomal binding site (RBS) or other elements that influence the promotor activity. Calculating the ideal level of promotor activity in a specific E. coli strain can be time consuming and needs to be optimized for each E. coli strain separately. The art also teaches that the psp promotor system is not very attractive for large-scale helper phage production due to the inflexibility of E. coli strains, the time consuming optimization and the significant low level of helper phage production.
One other significant problematic feature of all helper phage systems described is the occurrence of unwanted recombination events between the helper genome and the (helper-) plasmids. The problem that confronts investigators in the art is the fact that the g3 DNA sequences in the helper phages are homologous to the g3 sequences in the phage display vector and/or the helper phage plasmid. In many cases, this results in recombination between the two DNA strains and, therefore, loss of functionality of the library as a whole.
It is an object of the present invention to deal with problems and drawbacks known from the art as described above, concerning the generation of phage particles and helper phages, the use of helper phages in the production of phage display libraries, and the problems and drawbacks known for the identification of relevant binding molecules using such libraries.